Butanol is an important industrial chemical, useful as a fuel, fuel component and fuel additive, as a chemical solvent, feedstock in various chemical markets, such as the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.
Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A. Chem., 220:215-220 (2004)). These processes use starting materials derived from petrochemicals. The production of isobutanol from plant-derived raw materials would represent an advance in the art.
Isobutanol is produced biologically as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of the incomplete metabolism of amino acids by fungi. Isobutanol is specifically produced from catabolism of L-valine. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem., 273:25752-25756, 1998). U.S. Pat. Nos. 7,851,188 and 7,993,889 describe enzymatic pathways for the production of isobutanol in recombinant microorganisms.
An increase in the yield of C3-C5 alcohols from carbohydrates was shown when amino acids leucine, isoleucine, and/or valine were added to the growth medium as the nitrogen source (Internat'l. Patent Pub. No. WO 2005/040392). Similarly, Lilly et al. (FEMS Yeast Res., 6(5):726-743 (2006)) have demonstrated that the addition of a high concentration of valine to a fermentation medium increased Saccharomyces cerevisiae production of isobutanol, isobutyric acid, propanol and propionic acid concentrations.
Nako et al (Internat'l. Patent Pub. No. WO 2007/032522) note that amyl alcohol and/or isobutanol and/or isoamyl acetate levels in yeast used for the production of alcoholic beverages may be altered via manipulation of the BAT1 and BAT2 genes. See also, Chen, X. et al. (Biotechnology for Biofuels, 4:21 (2011)) and Yoshimoto, H., et al. (Appl. Microbial. Biotechnol., 59:501-508 (2002)).
U.S. Pat. Pub. No. 20100209986 describes means to produce metabolically-modified microorganisms useful for producing biofuels (e.g., isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol) from a suitable substrate. The methodology utilizes the organism's native metabolites in the amino acid biosynthetic pathway to produce biofuels by increasing flux towards the production of a 2-keto acid. The microorganism may comprise a 2-keto-acid decarboxylase, preferably selected from the group consisting of Pdc, Pdc1, Pdc5, Pdc6, Aro10, Thi3, KivD, and KdcA.
U.S. Pat. Pub. No. 20120045809 describes a recombinant eukaryotic microorganism capable of producing isobutanol from a carbon source, said recombinant eukaryotic microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant eukaryotic microorganism may overexpress a valine transaminase (encoded, e.g. by BAT1 or BAT2).
Enzymatic saccharification of cellulosic and/or lignocellulosic biomass may be employed to break down cellulose and hemicellulose to produce a hydrolysate containing sugars suitable for consumption by microorganisms (Lynd, et al., Microbiol. Mol. Biol. Rev., 66:506-577 (2002)). U.S. Pat. No. 5,231,017 describes a process for producing ethanol from raw materials, wherein a protease is utilized in combination with alpha-amylase and glucoamylase, to increase the rate and yield of ethanol production. Huo, Y.-X., et al. (Nature Biotechnol, 29(4):346-352 (2011)) suggest that a nitrogen-centric metabolic engineering strategy could be utilized to utilize proteins as feedstock for the production of biofuels.
Improvements and alternatives for the biosynthesis of butanol directly from plant-derived raw materials would improve economic viability and would represent an advance in the art.